Post-translational factors maintain the erythrocyte membrane skeleton, trigger its dissolution in senescent cells, and participate in its assembly. Similar interactions in non-erythroid cells participate in the process of membrane sorting and the maintenance of local receptor domains. The objective of this study is to understand the post-translational molecular mechanisms involved in the assembly, maintenance, and degradation of the membrane skeleton. Three approaches are used: i) purification and structural characterization of the proteins involved; ii) identification invitro of the sites of interaction between the proteins and the role of regulatory factors such as covalent phosphorylation or allosteric protein- protein interaction; and, iii) detailed analysis of the cytoskeletal changes accompanying certain hemolytic diseases, or differentiation in cultured leukemic cell lines. The study of hemolytic disease and culture cells allows a correlation of the interactions identified invitro with an invivo system. Problems of immediate interest include understanding the way covalent phosphorylation of spectrin alters the pathway of subunit assembly; the mechanism of allosteric stimulation of spectrin oligomer formation by ankyrin and protein 3; the way spectrin- actin binding is stimulated by protein 4.1; and the role of protein 4.9, calmodulin, and the 110/105 complex in the spectrin-actin-4.1 cytoskeletal structural unit. Also of concern is the influence of multisite phosphorylation of spectrin, ankyrin, protein 4.1, the 110/105 complex, and protein 3, and the kinases and phosphatases involved. Structural analysis entails limited proteolytic and chemical cleavage; peptide mapping; amino-acid and DNA sequencing. Active peptide fragments are prepared by limited proteolytic or chemical cleavage or by in vito synthesis from c-DNA clones. Small fragments re also prepared by solid phase peptide synthesis. Assays of in vitro function involve binding between radiolabeled components. Defects in the cytoskeleton are central to many hemolytic disorders. The paradigm offered by the red cell cytoskeleton also provides unique insights into the interactions of similar proteins in other cells. An understanding of the erythrocyte membrane skeleton will extend our knowledge of both erythroid and non- erythroid membrane cytoskeletal function.